An artificial nanocarrier with antioxidant function, its preparation method and application

Abstract

An artificial nanocarrier with antioxidant function, its preparation method, and its application, involving microbiology. It utilizes genetically engineered *Escherichia coli*. Escherichia coli :: ompA‑st Based on the cell membrane, it loads components via the SpyTag / SpyCatcher protein covalent linking system. Escherichia coli :: gshB‑sc Glutathione synthase B tagged with SpyCatcher was expressed. Two engineered bacterial strains, with preservation numbers GDMCC No. 67612 and 67613, were constructed, expressing the OmpA-st membrane protein and the GshB-sc fusion protein, respectively. Antioxidant artificial nanocarriers were obtained through disruption, purification, membrane fragment separation, covalent coupling, and ultracentrifugation. These carriers effectively scavenge reactive oxygen species, reduce ROS levels in recipient bacteria under benzo[a]pyrene stress, enhance cellular antioxidant capacity, and help cells resist oxidative stress.

Description

Technical Field

[0001] This invention relates to the field of microbiology, and in particular to an artificial nanocarrier with antioxidant function, its preparation method and application. Background Technology

[0002] Reactive oxygen species (ROS) are a class of common byproducts in the aerobic metabolism of organisms, including superoxide anions (O2). - ROS (Reactive Oxides) include hydroxyl radicals (·OH) and hydrogen peroxide (H2O2). Under normal physiological conditions, cells maintain a dynamic balance between ROS production and clearance. Low concentrations of ROS act as signaling molecules, participating in various life activities such as cell proliferation, differentiation, and immune responses. However, when the body is stimulated by internal or external environmental factors, such as ultraviolet radiation, environmental pollutant stress, or metabolic abnormalities, ROS can be produced explosively. Excessive ROS disrupts intracellular redox homeostasis, leading to oxidative stress. In this state, ROS indiscriminately attacks intracellular biomolecules, inducing lipid peroxidation, protein denaturation, and DNA damage, thereby damaging cell structure and function, inducing apoptosis, and is closely related to the development of various diseases such as aging, inflammation, neurodegenerative diseases, and cancer.

[0003] To mitigate the harmful effects of oxidative stress, various ROS scavenging strategies have been developed. Traditional methods primarily rely on the supplementation of exogenous antioxidants, such as small-molecule scavengers like vitamin C and vitamin E, or antioxidant enzymes like superoxide dismutase (SOD), catalase (CAT), and glutathione peroxidase (GPX). However, these methods have significant limitations. Small-molecule antioxidants typically lack targeting specificity, have low bioavailability, and are rapidly eliminated in vivo, making it difficult to maintain effective concentrations. While antioxidant enzymes exhibit high catalytic efficiency and specificity, as large protein molecules, they suffer from poor stability, susceptibility to protease degradation, weak cell membrane penetration, and the potential to induce immunogenicity, significantly limiting their effectiveness in practical applications. Therefore, developing an efficient, stable, and biocompatible delivery system to achieve precise delivery and targeted enhancement of antioxidant functional molecules is a pressing technical challenge.

[0004] Researchers have attempted to develop various materials science-based artificial nanocarrier systems. For example, they have encapsulated antioxidant enzymes using synthetic carriers such as liposomes, polymer nanoparticles (e.g., PLGA nanoparticles), mesoporous silica, and metal-organic frameworks (MOFs). While studies have shown that these artificial nanocarriers can protect enzymes from degradation and prolong their in vivo circulation time to some extent, they still face significant challenges in practical applications. First, synthetic materials often fail to mimic complex biological interface functions and lack intrinsic biological targeting capabilities. Second, polyethylene glycol (PEG) modification, widely used to evade immune clearance, has been shown to induce PEG antibodies in vivo, leading to the "accelerated blood clearance phenomenon" (ABC phenomenon), which conversely reduces the efficacy of repeated administration. Furthermore, the preparation of artificial carriers often involves organic solvents or severe physical shearing, which can easily cause denaturation and inactivation of the encapsulated enzyme protein. Moreover, the encapsulation efficiency for hydrophilic macromolecular proteins is typically low (often below 10%), making efficient delivery difficult. Therefore, finding a bio-based delivery system that combines high biocompatibility, high loading capacity, and natural targeting advantages is crucial to overcoming these bottlenecks.

[0005] To overcome the delivery barriers of free protein drugs, natural bacterial membrane vesicles (BMVs) have attracted significant attention due to their unique natural advantages. BMVs are nanoscale vesicles autonomously secreted by bacteria during their growth process. Their surfaces retain the natural membrane proteins and lipid components of the source strain, endowing them with excellent biocompatibility and homology targeting capabilities. However, the formation of bacterial membrane vesicles is a natural secretion process dependent on the metabolism of live bacteria. Limited by the complex intracellular transport mechanisms and compartmentalization barriers of bacteria, the efficiency of spontaneously encapsulating exogenous macromolecules (such as antioxidant enzymes) into bacterial membrane vesicles is extremely low and difficult to control artificially. Furthermore, the contents of naturally secreted bacterial membrane vesicles are extremely complex, often containing large amounts of uncontrollable endogenous nucleic acids, toxins, and other non-target proteins. This uncontrollable encapsulation not only severely dilutes the concentration of effective antioxidants but may also trigger unpredictable immunogenicity and biosafety risks.

[0006] To overcome the limitations of naturally occurring mechanisms and address the disadvantages of low encapsulation rates and uncontrollable cargo in natural vesicles, we aim to artificially construct nanocarriers using natural biomaterials (such as cell membranes) as raw materials. This artificial construction strategy, using natural cell membrane fragments as the outer shell material, cleverly preserves the abundant lipids and target receptor proteins on the original cell membrane, maintaining its natural biocompatibility advantage, while also enabling subsequent precise artificial intervention and purification of drug delivery in vitro. Furthermore, to improve cargo controllability and loading rate, a highly specific coupling system needs to be introduced. By adding corresponding protein linkers to the cell membrane and target protein, the target protein encapsulated during the physical reassembly of the nanocarrier can be enhanced, resulting in a higher loading rate.

[0007] Therefore, developing a nanocarrier that can effectively remove ROS and help recipient cells resist oxidative stress, and providing a new technical approach for its application in biomedicine, cosmetics, food and nutrition, has become an urgent technical problem to be solved in this field. Summary of the Invention

[0008] The purpose of this invention is to address the aforementioned problems in the prior art and provide a novel, genetically engineered artificial nanocarrier with antioxidant function, along with its preparation method and applications, to address the oxidative stress caused by biotoxic pollutants such as benzo[a]pyrene (BaP) on recipient bacteria. This method utilizes the SpyTag / SpyCatcher protein covalent linking system to efficiently and stably load glutathione synthase B (GshB) into engineered bacterial membrane vesicles. This system achieves precise and efficient coupling between the target protein and membrane fragments in vitro by adding corresponding protein linkers to the cell membrane (OmpA-ST) and the target protein (GshB-SC), respectively, utilizing their specific covalent reactions, thereby obtaining higher loading efficiency and stability.

[0009] To achieve the above objectives, the present invention adopts the following technical solution:

[0010] An artificial nanocarrier with antioxidant function, based on genetically engineered Escherichia coli Escherichia coli BL21:: ompA-st The cell membrane was loaded with SC-tagged glutathione synthase B to obtain an antioxidant-functional artificial nanocarrier.

[0011] The antioxidant-functional bacterial vesicles are made from genetically engineered Escherichia coli. Escherichia coli BL21:: ompA-st It was synthesized using cell membranes and purified SC-tagged glutathione synthase B as raw materials.

[0012] The genetically engineered E. coli Escherichia coli BL21:: ompA-st Import plasmid pET28a- ompA-st This strain was deposited on January 9, 2026, at the Guangdong Provincial Center for Microbial Culture Collection, China, with accession number GDMCC No. 67612. The deposit address is the Institute of Microbiology, Guangdong Academy of Sciences, 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou.

[0013] The plasmid pET28a- ompA-st The gene encoding the outer membrane protein OmpA (GCF_000767465.1 JI59_RS08840) and the SpyTag tag were inserted into the multiple cloning site of the pET28a backbone and linked with a 6×His purified tag, and constructed by homologous recombination.

[0014] The recipient cells are *Sphingomyelin-neostigmoides*. Novosphingobium pentaromativorans US6-1.

[0015] The purified SC-tagged glutathione synthase B was produced by a genetically engineered protein-expressing bacterium, *Escherichia coli*. Escherichia coli BL21:: gshB-sc After being broken down and purified, the strain was deposited on January 9, 2026, at the Guangdong Provincial Microbial Culture Collection Center, China, with accession number GDMCC No. 67613, located at the Institute of Microbiology, Guangdong Academy of Sciences, 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou.

[0016] The protein expression gene-engineered E. coli Escherichia coli BL21:: gshB-sc It was the introduction of plasmid pET28a- gshB-sc strains.

[0017] The plasmid pET28a- gshB-sc The gene was constructed using pET28a as a backbone, with the glutathione synthase B encoding gene (GCF_000767465.1 JI59_RS03695) and the SpyCatcher tag inserted into the multiple cloning site of the recipient bacterium genome, and linked with a 6×His purification tag, through homologous recombination.

[0018] A method for developing an antioxidant bacterial artificial nanocarrier includes the following steps:

[0019] (1) Culture and use IPTG-induced genetically engineered Escherichia coli Escherichia coli BL21:: ompA- st and Escherichia coli BL21:: gshB-scThe bacterial culture was centrifuged at high speed and the supernatant was discarded. The precipitate was collected and washed twice with PBS buffer to remove residual culture medium.

[0020] (2) Add 2 mL of hypotonic buffer to each of the above bacterial cells and resuspend them. Sonicate for 15 min, then centrifuge at high speed to remove the precipitate and obtain the supernatant. The centrifugation speed is 6,000 g, the centrifugation temperature is 4 ℃, and the centrifugation time is 20 min. Filter using a 0.45 μm filter to remove excess impurities to obtain the supernatant. Escherichia coli BL21:: ompA-st and Escherichia coli BL21:: gshB-sc The supernatant concentrate.

[0021] (3) The above Escherichia coli BL21:: gshB-sc After incubating the supernatant concentrate with His-tagged purified magnetic beads (4 °C, 4 h), the solution was eluted with imidazole buffer and concentrated through a 10 kDa ultrafiltration tube to obtain the purified SC-tagged glutathione synthase B protein solution.

[0022] (4) The above Escherichia coli BL21:: ompA-st After ultracentrifugation of the supernatant concentrate, the supernatant was discarded and the precipitate was collected. The ultracentrifugation rate was 142,000 g, the centrifugation temperature was 4 ℃, and the centrifugation time was 90 min.

[0023] (5) Resuspend the precipitate with an appropriate amount of hypertonic buffer, add purified SC-tagged glutathione synthase B solution, incubate on ice for 30 min, then perform ultracentrifugation and discard the supernatant. The ultracentrifugation rate is 488,000 g, the centrifugation temperature is 4 ℃, and the centrifugation time is 60 min. Resuspend with an appropriate amount of 10% glycerol to obtain the bacterial artificial nanocarrier with antioxidant function, and store at low temperature.

[0024] The culture medium for the genetically engineered Escherichia coli was LB (Luria-Bertan) medium.

[0025] A genetically engineered bacterial combination, the genetically engineered bacterial combination comprising Escherichia coli BL21:: ompA- st and Escherichia coli BL21:: gshB-sc The accession numbers are GDMCC No. 67612 and GDMCC No. 67613, respectively.

[0026] Application of antioxidant-functional artificial nanocarriers in the preparation of formulations that scavenge reactive oxygen species and resist cellular oxidative stress damage.

[0027] The specific application can be: artificial nanocarriers assist recipient cells in resisting oxidative stress generated during metabolism.

[0028] Compared with the prior art, the beneficial effects achieved by the technical solution of this invention are:

[0029] This invention uses genetically engineered bacterial cell membrane fragments as carrier materials, preserving the lipid composition and membrane protein structure of natural cell membranes. Compared to artificially synthesized carriers such as liposomes and PLGA, it exhibits better biocompatibility and lower immunogenicity, effectively avoiding accelerated blood clearance. This invention employs the SpyTag / SpyCatcher protein covalent coupling system, adding specific tags to both membrane proteins and target enzymes to achieve precise and efficient covalent binding of GshB-sc protein to cell membrane fragments. This overcomes the technical bottlenecks of low encapsulation rate and uncontrollable contents in natural membrane vesicles, significantly improving the loading efficiency and stability of antioxidant enzymes. Experiments show that the antioxidant artificial nanocarrier prepared by this invention maintains a stable particle size of 100-150 nm in an aqueous environment, with minimal long-term fluctuations in Zeta potential and PDI value, exhibiting excellent colloidal stability and monodispersity, effectively overcoming the defects of easy inactivation and degradation of free enzymes. Under benzo[a]pyrene (BaP) stress, the nanocarrier of the present invention can significantly reduce the intracellular ROS level of recipient bacteria and effectively maintain cell growth, demonstrating its clear and excellent function in responding to oxidative stress caused by environmental pollutants. It is expected to be applied in various practices to promote the antioxidant function of cells and provide new technical approaches for the fields of biomedicine, cosmetics, food and nutrition. Attached Figure Description

[0030] Figure 1 A schematic diagram illustrating the preparation method of an artificial nanocarrier with antioxidant function.

[0031] Figure 2 The images show scanning electron microscope (SEM) images of the antioxidant-functional artificial nanocarriers before and after ultracentrifugation; where A is an SEM image of cell membrane fragments before repolymerization; and B is an SEM image of the antioxidant-functional artificial nanocarriers after repolymerization.

[0032] Figure 3 The particle size change of artificial nanocarriers with antioxidant function after standing in Chenhai water for 6 days.

[0033] Figure 4 Changes in the zeta potential of an artificial nanocarrier with antioxidant function after standing in Chenhai water for 6 days.

[0034] Figure 5 The change in polydispersity index (PDI) of artificial nanocarriers with antioxidant function after standing in Chenhai water for 6 days.

[0035] Figure 6The growth of recipient bacteria after adding an antioxidant-functional artificial nanocarrier.

[0036] Figure 7 The effect of adding an antioxidant-functional artificial nanocarrier on the ROS levels of recipient bacterial cells under BaP stress. (Among them, This indicates that P < 0.05. This indicates that P < 0.01. (Indicates P < 0.0001) Detailed Implementation

[0037] To make the technical problems, technical solutions and beneficial effects of the present invention clearer and more understandable, the present invention will be further described in detail below with reference to the accompanying drawings and embodiments.

[0038] Example 1

[0039] This embodiment provides a method for preparing an antioxidant-functional artificial nanocarrier, the method comprising the following steps:

[0040] (1) Culture and use IPTG-induced genetically engineered Escherichia coli Escherichia coli BL21:: ompA- st and Escherichia coli BL21:: gshB-sc The bacterial culture was centrifuged at high speed and the supernatant was discarded. The precipitate was collected and washed twice with PBS buffer to remove residual culture medium.

[0041] (2) Add 2 mL of hypotonic buffer to each of the collected bacterial cells and resuspend them. Sonicate for 15 min, then centrifuge at high speed to remove the precipitate and obtain the supernatant. The centrifugation speed is 6,000 g, the centrifugation temperature is 4 ℃, and the centrifugation time is 20 min. Filter using a 0.45 μm filter to remove excess impurities to obtain the supernatant. Escherichia coli BL21:: ompA-st and Escherichia coli BL21:: gshB-sc The supernatant concentrate.

[0042] (3) The above Escherichia coli BL21:: gshB-sc After incubating the supernatant concentrate with His-tagged purified magnetic beads at 4°C for 4 h, it was eluted with imidazole buffer and concentrated using a 10 kDa ultrafiltration tube to obtain the purified SC-tagged glutathione synthase B solution.

[0043] (4) The above Escherichia coli BL21:: ompA-stThe supernatant concentrate was subjected to ultracentrifugation, after which the supernatant was discarded and the precipitate was collected. The ultracentrifugation rate was 142,000 g, the centrifugation temperature was 4 ℃, and the centrifugation time was 90 min.

[0044] (5) Resuspend the precipitate with an appropriate amount of hypertonic buffer, add purified SC-tagged glutathione synthase B solution, incubate on ice for 30 min, then perform ultracentrifugation and discard the supernatant. The ultracentrifugation rate is 488,000 g, the centrifugation temperature is 4 ℃, and the centrifugation time is 60 min. Resuspend in an appropriate amount of 10% glycerol to obtain an artificial nanocarrier with antioxidant function, and store at low temperature.

[0045] (6) Take 10 μL of the above supernatant concentrate and the antioxidant artificial nanocarrier respectively, observe their morphology using a transmission electron microscope, and at the same time use a nanoparticle size analyzer to determine the particle size, zeta potential and polydispersity index of the antioxidant artificial nanocarrier.

[0046] (7) Western blot verification showed that GshB-SC protein was specifically covalently bound to cell membrane fragments via SpyTag / SpyCatcher, with high coupling efficiency.

[0047] Figure 1 This paper provides a brief overview of the preparation method for antioxidant-functional artificial nanocarriers, clearly demonstrating the complete process from the construction and disruption of two genetically engineered bacteria to the coupling and repolymerization of cell membrane fragments with GshB-sc protein to form nanocarriers. Scanning electron micrographs of the antioxidant-functional artificial nanocarriers before and after ultracentrifugation are shown below. Figure 2 In this image, A is a scanning electron microscope image of the cell membrane fragments before repolymerization. It can be seen that the cell membrane before repolymerization consists of scattered, irregular fragments, without forming obvious spherical vesicle structures. Figure 2 B in the image is a scanning electron microscope image of the repolymerized antioxidant artificial nanocarrier. It can be seen that the repolymerized antioxidant artificial nanocarrier has a complete vesicle structure, indicating that the method can effectively drive the repolymerization of membrane fragments and stably form vesicles.

[0048] The physicochemical stability assessment of the antioxidant-functional artificial nanocarrier after standing in Chenhai water for 6 days is shown in [reference needed]. Figure 3 Nanoscale analysis showed that the antioxidant-functional artificial nanocarriers had a stable particle size distribution between 100 and 150 nm, and exhibited almost no significant change over time during a 6-day settling period, demonstrating good particle size stability. The zeta potential changes of the antioxidant-functional artificial nanocarriers after 6 days of settling in aged seawater are shown in [reference needed]. Figure 4The zeta potential of the antioxidant artificial nanocarrier remained stable between -12 and -15 mV during a 6-day settling period. Compared with natural vesicles and empty carriers, the potential fluctuation was smaller, indicating that it has superior colloidal stability in the aquatic environment and is less prone to aggregation or sedimentation. The polydispersity index (PDI) change of the antioxidant artificial nanocarrier after 6 days of settling in aged seawater is shown in [reference needed]. Figure 5 It can be seen that the polydispersity index (PDI) of the antioxidant artificial nanocarrier remained between 0.23 and 0.25 within 6 days, which was significantly lower than that of the natural vesicle and empty carrier groups, indicating that its particle size distribution was narrower and its uniformity was better, and it could maintain a stable monodisperse state even after long-term storage.

[0049] Example 2

[0050] Detecting the antioxidant function of artificial nanocarriers helps recipient bacteria cope with the oxidative stress caused by the highly toxic biopollutant benzo[a]pyrene.

[0051] The process of detecting reactive oxygen species levels in a culture system containing the pollutant benzo[a]pyrene, using an antioxidant artificial nanocarrier:

[0052] As described in Example 1, collect the antioxidant-functional artificial nanocarrier loaded with SC-tagged glutathione synthase B, and add the final OD... 600 The recipient bacterium, *Sphingosine monocytogenes*, had a concentration of 0.3. Novosphingobium pentaromativorans US6-1 was cultured at 30 °C in MM2 inorganic salt medium containing BaP at a final concentration of 10 ppm. Benzo[a]pyrene (BaP) was prepared into a 10000 ppm stock solution using dichloromethane and added to the medium at a volume ratio of 1:1000 to obtain a working solution with a final concentration of 10 ppm.

[0053] Cell growth and ROS levels were measured at 0 h, 12 h, 24 h, 48 h, 96 h, and 144 h of culture. The specific method was as follows: 1 mL of bacterial culture was centrifuged at 6,000 rpm for 8 min at 4 ℃, the supernatant was discarded, and the precipitate was washed twice with MM2 inorganic salt medium. The bacterial cells were resuspended in 1 mL of MM2, with 500 μL of the bacterial culture used for biomass measurement. The microplate reader was set to OD200. 600 Biomass was determined; the remaining 500 μL of bacterial culture was mixed with 0.5 μL of DCFH-DA probe, reacted for 15 min in the dark, centrifuged at 6,000 rpm for 8 min, the supernatant was discarded, and the bacterial cells were resuspended in 500 μL of MM2. 100 μL of sample was added to a 96-well plate, and the microplate reader parameters were set as follows: excitation wavelength 488 nm, emission wavelength 525 nm. Fluorescence values ​​were measured, and the measured fluorescence values ​​were compared with OD... 600The ratio represents the reactive oxygen species level of the sample strain.

[0054] Experimental results: See Figure 6 Under benzo[a]pyrene stress, the biomass (OD) of recipient bacteria in each group with added antioxidant artificial nanocarriers... 600 The growth rate was significantly higher than that of the "recipient bacteria" group and the "recipient bacteria + 20μg natural membrane vesicles" group, and the effect was dose-dependent. The growth advantage of the 40μg and 60μg groups was the most obvious, effectively maintaining the growth and metabolism of the recipient bacteria.

[0055] See Figure 7 Throughout the entire culture period, the intracellular ROS levels (relative fluorescence values) in all groups with added antioxidant artificial nanocarriers were significantly lower than those in the control group, and the ROS scavenging effect gradually increased with increasing carrier dosage. On day 6 and day 6 of culture, the intracellular ROS levels of the recipient bacteria were significantly reduced (…). Figure 7 This indicates that the nanocarrier can effectively remove excess reactive oxygen species in cells and significantly reduce oxidative stress damage.

[0056] In summary, this invention provides a method for an antioxidant-functional artificial nanocarrier and explores its application as a delivery carrier to deliver antioxidant-functional biomolecules to recipient cells to assist recipient cells in resisting oxidative stress generated during metabolism, thereby achieving precise delivery and targeted empowerment of antioxidant-functional molecules.

Claims

1. An artificial nanocarrier with antioxidant function, characterized in that: The artificial nanocarrier is based on genetically engineered Escherichia coli. Escherichia coli BL21:: ompA-st Cell membrane fragments, covalently coupled with SC-tagged glutathione synthase B, are artificial nanocarriers.

2. The artificial nanocarrier with antioxidant function as described in claim 1, characterized in that: The genetically engineered E. coli Escherichia coli BL21:: ompA-st To introduce plasmid pET28a- ompA-st The strain was deposited at the Guangdong Provincial Center for Microbial Culture Collection, China on January 9, 2026, with accession number GDMCC No. 67612.

3. The artificial nanocarrier with antioxidant function as described in claim 2, characterized in that: The plasmid pET28a- ompA-s t is constructed using pET28a as a backbone, with the gene encoding the outer membrane protein OmpA, GCF_000767465.1 JI59_RS08840, and the SpyTag tag inserted into its multiple cloning site in the recipient cell genome, and linked with a 6×His purified tag, through homologous recombination.

4. The artificial nanocarrier with antioxidant function as described in claim 3, characterized in that: The recipient cells are *Sphingomyelin-neostigmoides*. Novosphingobium pentaromativorans US6-1.

5. The artificial nanocarrier with antioxidant function as described in claim 1, characterized in that: The SC-tagged glutathione synthase B was produced by a genetically engineered E. coli strain. Escherichia coli BL21:: gshB-sc It was obtained by crushing and then purifying.

6. The artificial nanocarrier with antioxidant function as described in claim 5, characterized in that: The genetically engineered E. coli Escherichia coli BL21:: gshB-sc It is the imported plasmid pET28a- gshB-sc The strain was deposited at the Guangdong Provincial Center for Microbial Culture Collection, China on January 9, 2026, with accession number GDMCC No. 67613.

7. The artificial nanocarrier with antioxidant function as described in claim 6, characterized in that: The plasmid pET28a- gshB-sc It was constructed using pET28a as the backbone, with the glutathione synthase B encoding gene GCF_000767465.1 JI59_RS03695 and the SpyCatcher tag inserted into the multiple cloning site of the recipient bacterium genome, and linked with a 6×His purification tag, and then constructed through homologous recombination.

8. A method for preparing an artificial nanocarrier with antioxidant function as described in any one of claims 1 to 7, characterized in that, Includes the following steps: First, collect genetically engineered E. coli that have undergone induced culture. Escherichia coli BL21:: ompA-st and Escherichia coli BL21:: gshB-sc The culture medium residue was removed by centrifugation and washing; then the bacteria were sonicated in a hypotonic buffer, followed by centrifugation and filtration to remove cell debris and impurities; subsequently... Escherichia coli BL21:: gshB-sc The lysate supernatant was subjected to affinity purification to obtain GshB-SC protein. Escherichia coli BL21:: ompA-st The supernatant was subjected to ultracentrifugation to obtain membrane fragments containing OmpA-ST. The precipitate was resuspended in hypertonic buffer, and purified GshB-SC protein was added for ice bath incubation. After ultracentrifugation again, the supernatant was discarded, and the product was resuspended in 10% glycerol. The resulting product is the artificial nanocarrier with antioxidant function.

9. A genetically engineered bacterial combination, characterized in that: The combination contains genetically engineered bacteria. Escherichia coli BL21:: ompA-st and protein expression genetically engineered bacteria Escherichia coli BL21:: gshB-sc The accession numbers are GDMCC No. 67612 and GDMCC No. 67613, respectively.

10. The use of the antioxidant-functional artificial nanocarrier as described in any one of claims 1 to 7 in the preparation of formulations that scavenge reactive oxygen species and resist cellular oxidative stress damage.

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